{"id":604305,"date":"2023-02-03T16:52:23","date_gmt":"2023-02-03T22:52:23","guid":{"rendered":"https:\/\/news.sellorbuyhomefast.com\/index.php\/2023\/02\/03\/synapse-tuned-cars-enhance-immune-cell-anti-tumor-activity\/"},"modified":"2023-02-03T16:52:23","modified_gmt":"2023-02-03T22:52:23","slug":"synapse-tuned-cars-enhance-immune-cell-anti-tumor-activity","status":"publish","type":"post","link":"https:\/\/newsycanuse.com\/index.php\/2023\/02\/03\/synapse-tuned-cars-enhance-immune-cell-anti-tumor-activity\/","title":{"rendered":"Synapse-tuned CARs enhance immune cell anti-tumor activity"},"content":{"rendered":"\n
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Data availability<\/h2>\n

Data are available upon reasonable request. Source data<\/a> are provided with this paper.<\/p>\n<\/div>\n

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Code availability<\/h2>\n<\/div>\n
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References<\/h2>\n
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    \n

    Acknowledgements<\/h2>\n

    We would like to acknowledge the technical support from H. Houke, C. O\u2019Reilly and A. Chabot. This work was supported by the St. Jude Sumara Fellowship (P.C.), the American Lebanese Syrian Associated Charites (S.G.), the ChadTough Defeat DIPG Foundation (G.K.), National Institute of Neurological Disorders and Stroke grant R01NS121249 (G.K.), the Rally Foundation for Childhood Cancer Research (L.J.T.), the Garwood Postdoctoral Fellowship (J.I.V.) and National Institutes of Health (NIH)\/National Cancer Institute (NCI) grant P30 CA021765. Animal imaging was performed by the Center for In Vivo Imaging and Therapeutics, which is supported, in part, by NIH grants P01CA096832 and R50CA211481. Cellular images were acquired at the St. Jude Children\u02bcs Research Hospital Cell & Tissue Imaging Center, which is supported by St. Jude and NCI P30 CA021765. Gene editing of cell lines was performed by the Center for Advanced Genome Engineering (CAGE), which is supported, in part, by NCI P30 CA021765. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.<\/p>\n<\/div>\n

    \n

    Author information<\/h2>\n
    \n

    Authors and Affiliations<\/h3>\n
      \n
    1. \n

      Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA<\/p>\n

      Peter J. Chockley,\u00a0Jorge Ibanez-Vega,\u00a0Giedre Krenciute,\u00a0Lindsay J. Talbot\u00a0&\u00a0Stephen Gottschalk<\/p>\n<\/li>\n

    2. \n

      Department of Surgery, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA<\/p>\n

      Lindsay J. Talbot<\/p>\n<\/li>\n<\/ol>\n

      Contributions<\/h3>\n

      P.J.C. conceptualized the study, performed experiments, analyzed data and wrote the manuscript. J.I. performed confocal microscopy and analysis. G.K. provided DIPG007 and DIPG7c model setup for immune cell studies and acquired funding. L.J.T. developed and performed the halo assay. S.G. acquired funding, supervised the study and wrote the manuscript.<\/p>\n

      Corresponding author<\/h3>\n

      Correspondence to
      \n Peter J. Chockley<\/a>.<\/p>\n<\/div>\n<\/div>\n

      \n

      Ethics declarations<\/h2>\n
      \n

      Competing interests<\/h3>\n

      S.G. and P.J.C. have patent applications in the fields of NK and T cell and\/or gene therapy for cancer. S.G. has a research collaboration with TESSA Therapeutics, is a Data and Safety Monitoring Board member of Immatics and was on the scientific advisory board of Tidal. P.J.C. is a technical consultant for LUMICKS.<\/p>\n<\/p><\/div>\n<\/div>\n

      \n

      Peer review<\/h2>\n
      \n

      Peer review information<\/h3>\n

      Nature Biotechnology<\/i> thanks Dongfang Liu, Weidong Han and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.<\/p>\n<\/p><\/div>\n<\/div>\n

      \n

      Additional information<\/h2>\n

      Publisher\u2019s note<\/b> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.<\/p>\n<\/div>\n

      \n

      Extended data<\/h2>\n
      \n
      \n

      Extended Data Fig. 1 Primary NK cell transduction efficiency.<\/a><\/h3>\n
      \n

      (a<\/b>) Representative flow cytometry histogram plots detailing surface CAR expression. (b<\/b>) Quantified flow cytometry data showing percent CAR positive NK cells of various donors. UN: n\u2009=\u20099, CAR \u0394: n\u2009=\u20098, CAR: n\u2009=\u20099, CAR.PDZ: n\u2009=\u20096 donors, mean\u2009\u00b1\u2009SEM shown. (c<\/b>) Immunophenotype of NK cells via flow cytometry. n\u2009=\u20094 donors, mean\u2009\u00b1\u2009SEM shown.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 2 Scribble Polarization at 30\u2009minutes.<\/a><\/h3>\n
      \n

      (a<\/b>) Confocal images as prepared in (Fig. 1<\/a>) incubated for 30\u2009minutes with NK cells in various groups quantified in (b). White bars indicate 10microns. Immunolabelling of Scribble in red, CD3\u03b5 in green, and filamentous actin (F-actin) in white. (b<\/b>) Scribble polarization and accumulation at the immune synapse (IS). CAR \u0394; n\u2009=\u200913, CAR; n\u2009=\u20097, CAR.PDZmut: n\u2009=\u200914, CAR.PDZ n\u2009=\u20099, One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. mean\u2009\u00b1\u2009SEM shown of one donor.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 3 WASp Polarization at 15 and 30\u2009minutes.<\/a><\/h3>\n
      \n

      Confocal images as prepared in (Fig. 1<\/a>) incubated for 15 and 30\u2009minutes with NK cells in various groups. Quantified WASp polarization and accumulation at the immune synapse (IS). CAR \u0394; n\u2009=\u200934 and 25, CAR; n\u2009=\u200942 and 25, CAR.PDZ n\u2009=\u200919 and 39, for 15 and 30\u2009minutes, respectively. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. Statistical difference delineated by q\u2009<\u20090.01 *, q\u2009<\u20090.0001 ****; mean\u2009\u00b1\u2009SEM shown of one donor.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 4 Live NK cell imaging reveals lysosomal condensing and enhanced synapse formation with increased calcium Flux.<\/a><\/h3>\n
      \n

      Lysosomal coalescing from live cell imaging in Fig. 2g<\/a> EphA2 targeting CARs UN; n\u2009=\u200927, CAR\u0394; n\u2009=\u200948, CAR; n\u2009=\u200944, CAR.PDZ; n\u2009=\u200948 cells. Peak lysosome signal was measured from the first calcium flux peak in each condition. One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; mean\u2009\u00b1\u2009SEM shown of one donor.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 5 LM7 Avidity assessment with EphA2-targeting CARs.<\/a><\/h3>\n
      \n

      Normalized fold change of CAR NK cell binding compared to untransduced NK cells. Arrowed lines indicate the point of statistical difference at CAR.PDZ vs. CAR \u0394 at 268 pN, CAR.PDZ vs CAR at 343 pN, CAR.PDZ vs CAR.PDZmut at 363 pN which continued to 1000 pN indicated by dashed arrow lines. The only exception to this significance was from 650 to 738 pN for CAR.PDZ vs CAR.PDZmut. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; n\u2009=\u20091-3 donors, mean\u2009\u00b1\u2009SEM shown.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 6 B7-H3 CAR design with avidity, synapse, and calcium flux analyses.<\/a><\/h3>\n
      \n

      (a<\/b>) Chimeric antigen receptor design schemes. Antigen recognition domain (anti-B7-H3 scFv): goldenrod, hinge and transmembrane domains (CD8\u03b1H\/TM): grey, CD28 co-stimulatory domain: purple, CD3\u03b6 activation domain: blue, PDZbm scaffolding anchor domain: red. (b<\/b>) Example flow cytometry plot detailing B7-H3 CAR expression. (c<\/b>) Normalized fold change of CAR NK cell binding compared to untransduced NK cells. Bracketed line indicates the scale of statistical difference at CAR.PDZ vs. CAR \u0394 and CAR from 194 to 646 pN for both comparisons except for 205-215 pN. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; q\u2009<\u20090.05 *, <0.001 ***, n\u2009=\u20093 donors, mean\u2009\u00b1\u2009SEM shown. (d<\/b>) Immune synapse area quantification of B7-H3 CAR and CAR.PDZ NK cells (n\u2009=\u200911 and 11 cells) Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher\u2019s LSD test p\u2009<\u20090.05 * at minute 5 and 12 with area under the curve analysis. (e<\/b>) Calcium flux quantification of B7-H3 CAR and CAR.PDZ NK cells (n\u2009=\u200915 and 14 cells) with Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher\u2019s LSD test p\u2009<\u20090.05 * starting at minute 2; 1st<\/sup> peak AUC analysis with unpaired Student\u2019s t-Test, mean\u2009\u00b1\u2009SEM shown of one donor.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 7 A549 and LM7 tumor rechallenge rejection.<\/a><\/h3>\n
      \n

      (a<\/b>) A549 tumor rechallenge timeline with identical initial cancer cell numbers. Indicated tumor volumes from palpable nodules overtime. (b<\/b>) Intravital imaging of LM7 rechallenge with identical initial cancer cell numbers in complete responder mice. Color scale 1e6<\/sup> to 1e7<\/sup> of total photon flux(p\/s). (c<\/b>) Tumor flux values of weekly measurements.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 8 CAR T cell phenotyping and cytokine production.<\/a><\/h3>\n
      \n

      (a<\/b>) Chimeric antigen receptor design schemes. Antigen recognition domain (anti-B7-H3 scFv): goldenrod, hinge and transmembrane domains (CD8\u03b1H\/TM): grey, CD28 co-stimulatory domain: purple, CD3\u03b6 activation domain: blue, PDZbm scaffolding anchor domain: red. (b<\/b>) Transduction efficiencies of various CAR constructs in T cells CAR \u0394; n\u2009=\u20097, CAR; n\u2009=\u20098, CAR.PDZ n\u2009=\u20095 donors mean\u2009\u00b1\u2009SEM shown. (c<\/b>) CD4\/8\u2009T cell analysis post transduction at Day 5-6, n\u2009=\u20092 donors mean\u2009\u00b1\u2009SEM shown. (d<\/b>) Immunophenotype of CD4 CAR T cells Day 5-6 and longitudinally Day 12-13, n\u2009=\u20092 donors mean\u2009\u00b1\u2009SEM shown. (e<\/b>) Immunophenotype of CD8 CAR T cells Day 5-6 and longitudinally Day 12-13, n\u2009=\u20092 donors mean\u2009\u00b1\u2009SEM shown. (f<\/b>) B7-H3 CAR T cells were co-cultured with A549, LM7, and 143b cancer cells lines at a 2:1 ratio for 24\u2009hours for n\u2009=\u20093 experiments. Supernatant was collected and multiplex cytokine assessment was performed. One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR mean\u2009\u00b1\u2009SEM.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 9 B7-H3 CAR T cell synapse and calcium flux analyses.<\/a><\/h3>\n
      \n

      (a<\/b>) Immune synapse area quantification of B7-H3 CAR and CAR.PDZ T cells (n\u2009=\u200912 and 9 cells) co-cultured with LM7 cells with Area Under the Curve analysis. (b<\/b>) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cell (n\u2009=\u200916 and 22 cells) co-cultured with LM7 cells with Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher\u2019s LSD test p\u2009<\u20090.0001 **** at minute 4; 1st<\/sup> peak AUC analysis. mean\u2009\u00b1\u2009SEM shown of one donor. (c<\/b>) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cells (n\u2009=\u200942 and 15 cells) co-cultured with U87 cells. Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher\u2019s LSD test p\u2009<\u20090.0001 **** starting at minute 1; 1st<\/sup> peak AUC analysis with unpaired Student\u2019s t-Test. mean\u2009\u00b1\u2009SEM shown of one donor. (d<\/b>) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cells (n\u2009=\u200922 and 31 cells) co-cultured with DIPG007 cells. Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher\u2019s LSD test p\u2009<\u20090.0001 **** starting at minute 1; 1st<\/sup> peak AUC analysis with unpaired Student\u2019s t-Test. mean\u2009\u00b1\u2009SEM shown of one donor.<\/p>\n

      \n Source data<\/a>\n <\/p>\n<\/div>\n<\/div>\n

      \n

      Extended Data Fig. 10 B7-H3 expression on tumor cells.<\/a><\/h3>\n

      143B, U87, LM7, DIPG7c, and DIPG007 tumor cells were analyzed for B7-H3 expression. Black histograms indicate Isotype controls and red histograms are immunolabeled cells. Each sample is 100% positive and the indicated gMFI values are delineated.<\/p>\n<\/div>\n<\/div>\n<\/div>\n

      \n

      Supplementary information<\/h2>\n<\/div>\n
      \n

      Source data<\/h2>\n<\/div>\n